PROJECT SUMMARY The guidance of axons to their correct targets is an important step in nervous system wiring, but the molecular and cellular mechanisms of axon guidance are still not completely understood. The goal of the proposed research is to elucidate the functions of a new axon guidance cue, NELL2, and its receptor Robo3 in neural development and to define the molecular mechanisms mediating NELL2-Robo3 signaling. While the receptors Robo1 and Robo2 mediate axon repulsion by guidance molecules of the Slit family, the divergent Robo family member Robo3 does not bind Slits but instead silences Slit-Robo1/2 signaling. Furthermore, Robo3 indirectly potentiates axonal attraction by Netrin-1, mediated by the Netrin receptor DCC. We recently identified a novel guidance cue, NELL2, as a repulsive ligand for the previously orphan receptor Robo3. We generated NELL2 knockout mice and found that NELL2/Robo3-mediated repulsion guides commissural axons in the embryonic spinal cord. Our findings define a novel NELL2-Robo3 signaling pathway for axon guidance and raise the possibility that it wires neural circuits throughout the nervous system. To test this idea, we will analyze the expression of NELL2 and Robo3 during neural development by several means, including genetic reporter lines. Neuronal populations that express Robo3 or project axons close to sites of NELL2 expression will be examined for NELL2 responses in vitro. In these axon guidance assays, Robo3-dependence will be tested using neurons from Robo3 mutant mice. To determine the in vivo roles of NELL2 and Robo3 in wiring neuronal populations of interest, axonal trajectories in NELL2- and Robo3-deficient mice will be analyzed by in toto imaging of the embryonic nervous system and by several complementary approaches. Based on preliminary expression data, initial studies will focus on retinal ganglion cell axon guidance. Our results from commissural neurons show that Robo3 function in axon guidance is three- fold, as it inhibits Slit-induced repulsion through Robo1/2, potentiates Netrin-1 attraction through DCC, and mediates repulsion from its own ligand NELL2. It is unclear which intracellular signaling pathways are engaged downstream of NELL2 and how Robo3 is able to perform its multiple functions simultaneously. To address these questions, we will test if Slit silencing, Netrin-1 attraction, and NELL2 repulsion can be separated in Robo3 structure-function analyses in vitro and in vivo. Using candidate and unbiased biochemical approaches, we will identify novel NELL2 receptors and downstream mediators of Robo3 signaling and determine their roles in axon guidance in vitro and neural circuit formation in vivo. Our work is expected to provide important insights into the functions of NELL2 and Robo3 in brain wiring. It will also help elucidate how different axon guidance signaling pathways interact within the same neuron, a fundamental question in neural development that is still poorly understood. By defining mechanisms of axon pathfinding, the proposed work has the potential to inform therapeutic approaches aimed at restoring damaged axonal connections after physical injury or onset of neurodegenerative disease. Furthermore, it can help define the mechanisms underlying the etiology of diseases that result from nervous system mis-wiring.